|Protein name||Type-2 restriction enzyme PvuII||type II site-specific deoxyribonucleasetype II restriction enzyme|
|Synonyms||R.PvuIIEC 126.96.36.199Type II restriction enzyme PvuIIEndonuclease PvuII|
|Activity||Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.|
|Cofactor||Binds 2 magnesium ions per subunit.|
|Type||divalent metal (Ca2+, Mg2+)||nucleic acids||H2O||nucleic acids,phosphate group/phosphate ion||nucleic acids|
|1eyuA||Unbound||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA)||Unbound||Unbound|
|1eyuB||Unbound||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA)||Unbound||Unbound|
|1f0oA||Analogue:2x_CA||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA)||Unbound||Unbound|
|1f0oB||Analogue:2x_CA||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA)||Unbound||Unbound|
|1pviA||Unbound||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA)||Unbound||Unbound|
|1pviB||Unbound||Bound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA)||Unbound||Unbound|
|2pviA||Unbound||Analogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C(chain D:double stranded iodinated cognate DNA)||Unbound||Unbound|
|2pviB||Unbound||Analogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C(chain C:double stranded iodinated cognate DNA)||Unbound||Unbound|
|References for Catalytic Mechanism|
|References||Sections||No. of steps in catalysis|
|||Fig.8, Fig.11, p.12-17||2|
|Authors||Cheng X, Balendiran K, Schildkraut I, Anderson JE|
|Title||Structure of PvuII endonuclease with cognate DNA.|
|Comments||X-ray crystallography (2.4 Angstroms)|
|Journal||Nat Struct Biol|
|Authors||Athanasiadis A, Vlassi M, Kotsifaki D, Tucker PA, Wilson KS, Kokkinidis M|
|Title||Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV.|
|Authors||Jeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A|
|Title||Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.|
|Journal||Eur J Biochem|
|Authors||Pingoud A, Jeltsch A|
|Title||Recognition and cleavage of DNA by type-II restriction endonucleases.|
|Journal||J Biol Chem|
|Authors||Nastri HG, Evans PD, Walker IH, Riggs PD|
|Title||Catalytic and DNA binding properties of PvuII restriction endonuclease mutants.|
|Comments||X-ray crystallography (1.9 Angstroms)|
|Authors||Horton JR, Bonventre J, Cheng X|
|Title||How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?|
|Comments||X-ray crystallography (2.15 Angstroms)|
|Journal||Proc Natl Acad Sci U S A|
|Authors||Horton NC, Newberry KJ, Perona JJ|
|Title||Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.|
|Journal||J Mol Biol|
|Authors||Horton JR, Nastri HG, Riggs PD, Cheng X|
|Title||Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.|
|Journal||J Mol Biol|
|Authors||Horton JR, Cheng X|
|Title||PvuII endonuclease contains two calcium ions in active sites.|
|Authors||Dupureur CM, Conlan LH|
|Title||A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.|
|Authors||Dall'Acqua W, Carter P|
|Title||Substrate-assisted catalysis: molecular basis and biological significance.|
|Journal||J Mol Biol|
|Authors||Simoncsits A, Tjornhammar ML, Rasko T, Kiss A, Pongor S|
|Title||Covalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.|
|Authors||Dominguez MA Jr, Thornton KC, Melendez MG, Dupureur CM|
|Title||Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease.|
|Journal||J Mol Biol|
|Authors||Rauch C, Trieb M, Flader W, Wellenzohn B, Winger RH, Mayer E, Hallbrucker A, Liedl KR|
|Title||PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA.|
|Authors||Conlan LH, Dupureur CM|
|Title||Multiple metal ions drive DNA association by PvuII endonuclease.|
|This enzyme belongs to the type II restriction endonucleases.|
According to the paper , cleavage of DNA by restriction endonucleases yields 3'-OH and 5'-phosphate ends, where hydrolysis of the phosphodiester bonds by EcoRI and EcoRV occurs with inversion of configuration at the phosphorous atom, suggesting an attack of a water molecule in line with the 3'-OH leaving group. In general, hydrolysis of phosphodiester bonds requires three functional entities as follows :
(1) A general base that activates the attacking nucleophile,
(2) A Lewis acid that stabilizes the extra negative charge in the pentacovalent transition state,
(3) An acid that protonates or stabilizes the leaving group.
The literature  also described the two possible catalytic mechanisms, the substrate-assisted catalysis model and the two-metal-ion mechanism, as described in the following paragraph. However, this paper supported the substrate-assisted catalysis model more favorably than the two-metal-ion mechanism.
(1) Substrate-assisted catalysis model: The attacking water molecule is oriented and deprotonated by the next phosphate group 3' to the scissile phosphate. The negative charge of the transition state could be stablized by the Mg2+ ion and the semi-conserved lysine. The metal ion is bound by the two conserved acidc amino acid residues. The 3'-O- leaving group is protonated by a Mg2+-bound water .
(2) Two-metal-ion mechanism: A metal ion bound at one site is responsible for charge neutralization at the scissile phosphate. The attacking water is considered to be part of the hydration sphere of a metal ion bound at the second site .
The literature  described two possible catalytic mechanisms for type II restriction endonucleases. Both mechanisms involve two acidic amino acids (Asp58 and Glu68) and one basic amino acid (Lys70). In the enzyme-DNA complex, a binding site for a Mg2+ ion is formed by the sidechains of Asp58 and Glu68. The amino group of Lys70 stabilizes the transition state and acts as a Lewis acid in the reaction.
(1) Substrate-assisted, one metal ion mechanism: The phosphate group of the adjacent T(+2) should act as a general base where the attacking water molecule "A" is deprotonated.
(2) Two metal ion mechanism: A second Mg2+ ion should be coordinated to Glu55/Asp58 site and would neutralize the charge of the scissile phosphate of C(+1). However, Glu55 is not crucial in the metal coordination; it does not exclude the possiblity that a second Mg2+ could be coordinated in the same vicinity via water molecules, protein mainchain atoms, and/or the DNA phosphate backbone.
The literature  also suggested another possible mechanism, three-metal ion mechanism for type II restriction endonucleases from the structural data of EcoRV, as follows:
A metal ion at site I ligates through water to the 3'-phosphate. A second inner-sphere water molecule on this metal dissociates to provide the attacking hydroxide ion, and this dissociation is aided by the immediately adjacent lysine residue, corresponding to Lys70 in this enzyme. The metal at site III provides stabilization of the incipient negative charge as the transtion state develops. An inner-sphere water on this metal is located within hydrogen-bonding distance of the leaving 3'-oxygen. Thus, the site III metal is suggested to be operative in lowering the pKa of this water, so that it may dissociate to immediately protonate the leaving anion . The site II metal is purely structural .
Crystal structures of these type II endonucleases, EcoRV, EcoRI and this enzyme, PvuII bound to DNA show that the relative positions of the scissile and adjacent 3'-phosphates are conserved. Therefore, the two metal ions bound in site I and site III may have similar functions in each of these enzymes .
More recently, several papers including  supported the substrate-assisted mechanism for this enzyme and related enzymes (type II restriction enzymes), ruling out the two-metal-ion mechanism. Thus, we concluded that this enzyme adopts the substrate-assisted mechanism with only one metal ion for catalysis (see EcoRV; S00404 in EzCatDB).
Considering the structure of 1f0o and in-line attack by water on the scissile phosphoric ester bond, the substrate-assisted mechanism seems to be more likely, and the reaction probably proceeds as follows:
(1) Substrate-assisted Water activation by the 3'-phosphate group of adjacent nucleotide of the DNA (distance between the base-phosphate oxygen and the water, 2.36 A, and that between the water and calcium ion, 2.79 A, in enzyme chain B). This activated water is stabilized by lys70 (distance 3.39 A in enzyme chain B).
(2) The activated water makes a nucleophilic attack on the phosphorus atom in line with the P-O3' bond. (distance 3.39 A in enzyme chain B)
(3) Transition-state is stabilized by (Lys70 and) magnesium ion (distance 4.30 A with lys70, and 2.32 A and 2.60 A with the two calcium ions, analogues of magnesium ions, in enzyme chain B)
(4) Another water, which is bound to magnesium ion and Asp58, acts as a general acid to protonate the leaving O3' atom. (There is no catalytic acid in enzyme chain B for DNA chain C, whereas distance between O3' & water 3.17A, that between calcium and water, 3.46 A, and that between Asp58 and water, 2.55 A for DNA chain D and protein chain A) (This water also interacts with the phosphate oxygen (distance 3.39 A) in chain A.)